Toll-Like Receptor 4 Expression and Activity is Elevated in Human Diabetic Retinas and in Retinal Cells Exposed to Elevated Glucose Conditions

Abstract

Purpose: : We have previously shown that the expression of toll-like receptor 4 (TLR4) on circulating immunocytes of diabetic patients positively correlates with clinical signs of diabetic retinopathy (DR). Here we measured TLR4 expression and activity in diabetic retinas or in isolated retinal cells exposed to elevated glucose conditions.

Methods: : Human post-mortem retinas from diabetic donors (Type1 and Type 2) and non diabetic (control) donors were obtained from Georgia Eye Bank. Isolated retinal cells (retinal endothelial cells, microglia and Muller cells) were exposed to high glucose condition (HG= 25mM D-glucose) or the osmotic control (LG= 25mM L-glucose) for different times (12, 24, 48 and 72 hours). Western blotting analysis and quantitative RT-PCR was used to determine the expression of TLR4 at protein and mRNA level, respectively. TLR4 activity was assessed by measuring phosphorylation of MyD88 and IRAK-1 by immunoblotting using anti-phospho-specific antibodies.

Results: : Western analysis revealed that in human diabetic retinas TLR4 expression was significantly increased as compared to control donor retinas. MyD88 and IRAK-1 serine phosphorylation was also augmented. Exposure of isolated retinal cells to HG stimulated TLR4 expression, at protein and mRNA levels, effect that peaked at 24 hours. HG also promoted TLR4 activation as shown by increased phosphorylation of the downstream effectors MyD88 and IRAK-1. All these effects were not induced by exposure of the cells to the osmotic control (LG).

Conclusions: : Given TLR4 critical role in adaptive immunity and induction of inflammatory processes, our data directly implicate TLR4 in DR pathogenesis. Our results, further, demonstrate that activation of TLR4 signaling events in circulating immunocytes of diabetic patients mirrors retinal expression and activity of TLR4, thus supporting its monitoring as possible diagnostic tool for DR progression.

Keywords: diabetic retinopathy • inflammation • diabetes